This invention relates to a family of reagents which have affinity for fibrin. The reagents comprise a moiety defining a fibrin binding site linked to a biologically active domain having fibrinolytic or therapeutic activity, or a property which enables imaging of intravascular thrombi. More particularly, the invention relates to a method of imparting to injectable agents an affinity for fibrin.
Spontaneous formation of blood clots and their subsequent dissolution by the coagulation and fibrinolytic systems are important functions in the body's natural healing process. However, the formation of intravascular clots is the cause of many diseased conditions such as deep vein thrombosis, stroke, and myocardial infarction. Intravascular anticoagulant therapy has been used successfully to prevent the formation of clots, but treatment options are limited after a thrombus has formed and is lodged within the body.
Currently, there are a number of drugs under development or in clinical testing which may be useful as intravascular fibrinolytic agents. For example, recombinant tPA, urokinase, prourokinase, and streptokinase have been, now are, or will soon be offered commercially for this purpose. TPA is a serine protease which catalyzes the conversion of the inactive protein plasminogen to plasmin. Plasmin then dissolves the fibrin clot. TPA can bind specifically to fibrin and its proteolytic action is thereby at least theoretically limited to the location of the clot. Prourokinase is fibrin selective. Urokinase and streptokinase, on the other hand, do not exhibit this fibrin binding specificity or selectivity. Administration of these reagents can cause significant peripheral bleeding.
While the successful management of these diseased states will be aided when development of the therapeutic agents discussed above is complete, there is nevertheless a need for therapeutic and diagnostic agents which selectively bind to intravascular thrombi. In addition to fibrinolytic agents, reagents useful for imaging clots which would permit the physician to determine their precise location would be valuable. Also, analgesic and therapeutic agents which could be targeted to the sight of a wound where clotting naturally occurs would have clinical value.
Monoclonal antibodies potentially can provide a moiety which specifically binds to an epitope of fibrin or another constituent of a thrombus. See Haber et al, EPO 187,658. The state of the art is such that coupling biochemically active agents to an antibody so that the agent retains its biological activity and the antibody retains its specificity can be achieved readily. A monoclonal antibody tagged, for example, with a gamma emitting radioactive atom, theoretically could be administered to image a thrombus. See Childs et al, J. Nucl. Med. V. 26, No. 3, pp 293, and Rosebrough, Radiology, 1985, 156:515. However, administration of murine and rat monoclonals to humans has potentially serious immunological side effects. Monoclonals of human origin which might be less antigenic are notoriously difficult to produce.
U.S. Pat. No. 4,312,942 discloses a reagent for the detection of fission products of fibrinogen and/or fibrin which comprises, as an active component, a suspension of a clumping factor-positive microorganism extract from Staphylococcus aureus, especially strain K807. It is known that Protein A from Staphylococcus aureus binds with the F.sub.c fragment of human immunoglobulin (see, e.g., U.S. Pat. No. 4,478,914). Howiger et al, in Ann NY Acad. Sci., 1983, Vol. 408, pgs. 521-535, disclose that fibrinogen interacts with several types of cells including Staphylococcus aureus. The interaction is said to involve the binding of fibrinogen to specific cell receptors resulting in clumping of the bacteria. This article reviews research directed to the interaction of fibrinogen with procaryotic cells. Kolawole, in J. Hyg., (Camb.) 1984, Vol. 92, Pgs. 183-192, disclose the results of a study of the mechanism by which Staphylococcus aureus strains assume increased resistance to polymorph bactericidins if grown in plasma. The author reports that one of the mechanisms of acquiring resistance appears to involve an interaction between Staphylococcus aureus and fibrinogen, resulting in the deposition of fibrin or fibrin derivatives on the bacterial surface. The author speculates that the presence of fibrin on the cell surface could result from interactions between plasma proteins and Staphylococcus aureus factors such as clumping factor, free coagulase, or protein A, citing Forsgren and Sjoquist, Protein A from Staphylococcus aureus, J. Immuno. V., 97, pgs. 822-827.
Also, various radioactive atoms have been coupled with tPA, Fragment E.sub.1 from the plasmic digestion of cross-linked fibrin, and human platelets for use as imaging agents. See, e.g., Knight et al, Radiology, 1985, V. 156, pp 509-514; Knight et al, The Journal of Nuclear Medicine, Vol. 19, No. 8, p 891; and Krohn et al, Seminars in Nuclear Medicine, Vol 8, No. 3, July, 1977. In these studies I-123, In-111, Br-77, Tc-99, and I-131 have been linked to materials having fibrin affinity by various conventional methods.